Neuroprotective peptide

ABSTRACT

A peptide which has a neuroprotective action, a neuroprotective drug and neuropathic treatment containing the peptide, and a preventive or alleviative pharmaceutical composition. The peptide comprises proline-rich protein 4 having the sequence of SEQ ID NO:1 or a partial sequence thereof, or a modified sequence thereof, which has a neuroprotective action.

FIELD

The present invention relates to a peptide which has a neuroprotectiveaction, a neuroprotective agent comprising the peptide and apharmaceutical composition for treating or preventing nerve damage,comprising the peptide.

BACKGROUND

Nerve cells are cells that constitute the nervous system, which can bebroadly divided into the central nervous system and the peripheralnervous system. Nerve cells are prone to be damaged by external factorssuch as a cerebrovascular injury such as a stroke, a brain infarction,etc.; or infernal factors such as an accumulation of abnormal proteins,oxidant stress, inflammation, etc., while their regenerative capacity islow. Thus, once nerve cells are damaged, those damaged nerve cells causea marked reduction in the QOL of a patient. Neurodegenerative disordersthat involve degeneration and loss of nerves of the central nervoussystem, include neurodegenerative diseases such as Alzheimer's disease,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, multiple sclerosis, etc. as well as degenerative diseases ofthe optic nerve such as glaucoma, etc.; and neurosensory degenerativediseases such as neural hearing loss, etc.

With developments in neuro science, various neuroprotective factors havebeen discovered, and they are expected to developed as a drag forpreventing or treating nerve damage. While agents that reduce freeradicals or excitatory amino acids and agents that can protect and/orrepair nerve cells (e.g., immunophilin ligands such as neurotrophicfactors and immunosuppressants) have been found to have aneuroprotective action, it has also been found that biological proteins,such as pituitary adenylate cyclase-activating polypeptide (PACAP), CD44and human brain carboxypeptidase B (HBCPB), have a neuroprotectiveaction. (PTL 1 and 2)

Human tears contain numerous proteins such as lysozymes, lactoferrins,etc., which serve to maintain the homeostasis of the corneal surface.Moreover, tears also contain degradants of proteins, and it is expectedthat such peptide fragments have various actions. Proline-rich protein 4(PRP4) known to be present in tears is a protein having a fall aminoacid length of 134 residues. Since it has an antibacterial action, it issuggested that it acts as a protective mechanism against pathogens inmucous tissues (NPL 1).

CITATION LIST Patent Literature

[PTL 1] JP 2014-510101

[PTL 2] JP 2012-232952

Non-Patent Literature

[NPL 1] Proteomics 2004, 4, 3953-3959

SUMMARY Technical Problem

The purpose of the present invention is to provide a novel peptide whichhas a neuroprotective action, and a composition for treating orpreventing nerve damage comprising the peptide.

Solution to Problem

The present inventors discovered that proline-rich protein 4 (PRP4) andpeptide fragments isolated therefrom, have a neuroprotective action, andthereby completed the present invention.

The present invention relates to the following:

[1] A peptide consisting of the amino acid sequence of QQRPP (SEQ IDNO:3) or a sequence derived therefrom by addition of one or two aminoacids to N-terminal, C-terminal or both terminals thereof, wherein thepeptide has a neuroprotective action.

[2] The peptide according to item 1, wherein the peptide consists of asequence wherein one or two amino acids are added to N-terminal.

[3] The peptide according to item 1 or 2, wherein the amino acids addedto N-terminal are glutamine-asparagine- or asparagine-, and the aminoacids added to C-terminal are -glutamine-arginine or -glutamine.

[4] A peptide consisting of a sequence of QQRPP (SEQ ID NO:3).

[5] A neuroprotective agent comprising the peptide according to any oneof items 1-4.

[6] A pharmaceutical composition for treating or preventing nerve damagecomprising any one of items 1-4.

[7] A method of treating or preventing nerve damage, comprisingadministering the peptide according to any one of items 1-4, or theneuroprotective agent according to item 5, to a subject having nervedamage.

[8] The peptide according to any one of items 1 to 4, for use intreating or preventing nerve damage or in nerve protection.

[9] Use of the peptide according to any one of items 1 to 4 or theneuroprotective agent according to item 5 for the manufacture of atherapeutic agent or a preventive agent for nerve damage.

[10] The pharmaceutical composition according to item 6, the methodaccording to item 7, the peptide according to item 8, or the useaccording to item 9, wherein the nerve damage is a neurodegenerativedisease or a cerebrovascular disorder.

[11] The pharmaceutical composition, method, peptide or use according toitem 10, wherein the neurodegenerative disease is selected from thegroup consisting of dementia, Parkinson's disease, spinocerebellardegeneration, Creutzfeldt-Jakob disease, Alzheimer's disease,Huntington's chorea, multiple sclerosis, mad cow disease, spinalprogressive muscular atrophy, amyotrophic lateral sclerosis, spinal andbulbar muscular atrophy, glaucoma, retinal pigmentary degeneration,age-related macular degeneration, diabetic retinopathy, hearing loss andepilepsy.[12] The method according to item 7, the peptide according to item 8, orthe use according to item 9 for treating or preventing nerve damagethrough nerve protection.

Advantageous Effects of Invention

A peptide of the present invention inhibits the death of nerve cells,and consequently has a neuroprotective action. Therefore, a peptideaccording to the present invention can be used in the treatment orprevention of nerve damage. Moreover, since a peptide according to thepresent invention is a fragment of protein present in human tears, itcan be developed as a medicine with low side effects because it has lowtoxicity and low immunogenicity when used in a living body.

DESCRIPTION OF EMBODIMENTS

The present invention relates to a peptide consisting of proline-richprotein 4 having the sequence of SEQ ID NO: 1 or a partial sequencethereof, or a modified sequence thereof, which has a neuroprotectiveaction. In a preferred aspect, it is preferable that such a peptide bean isolated and/or purified peptide. The phrase “isolated and/orpurified peptide” is intended to refer to a substantially isolatedpeptide of interest and to distinguish it from a peptide merely existingas a mixture in an unseparated stale, such as is a body fluid or in amixture following protease treatment for analysis.

TABLE 1 Name Sequence PRP4 MLLVLLSVVLLALSSAQSTDNDVNYEDFTFTIPDVEDSSQRPDQGPQRPPPEGLLPRPPGDSGNQDDGPQQRPPKPGGHHRHPPPPPFQNQQRPPQRGHRQLSLPRFPSVSLQEASSFFRRDRPARHPQEQPLW (SEQ ID NO: 1)

In one situation, in one aspect of the present invention, a peptide ofthe present invention is a peptide having the sequence of SEQ ID NO: 2or a partial sequence thereof, or a peptide consisting of a modifiedsequence thereof, and having an amino acid length of 50 to 200 residues.It is more desirable that such a peptide comprise, for example, 80 to150 amino acid residues, it is particularly desirable that it comprise90 to 140 amino acid residues, and it is even more desirable that itcomprise 100 to 140 amino acid residues. Such a long-chain peptide canexhibit neuroprotective action in that state or can produce a peptide ofthe present invention through the action of protease in the living bodyto exhibit neuroprotective action. As an example, a peptide (PRIM(human) recombinant protein: catalog number H0001127), which is marketedby Abnova Corporation, comprising die amino acid sequence of position 17to position 116 (SEQ ID NO: 2) of proline-rich protein 4 can be used.

TABLE 2 Name Sequence PRP4 QSTDNDVNYEDFTFTIPDVEDSSQRPDQGPQRPPPEGLL(posi- PRPPGDSGNQDDGPQQRPPKPGGHHRHPPPPPFQNQQRP tion 17PQRGHRQLSLPRFPSVSLQEAS to posi- (SEQ ID NO: 2) tion 116)

In another aspect of the present invention, the present inventionrelates to a peptide comprising the amino acid sequence of position 17to position 116 of an amino acid sequence of proline-rich protein 4 (SEQID NO: 1), having a neuroprotective action. Such a peptide may have asequence to which any amino acids are added to the N-terminal andC-terminal of the amino acid sequence of position 17 to position 116(SEQ ID NO: 2) of proline-rich protein 4, but it is desirable that thepeptide have amino acids corresponding to the full-length sequence (SEQID NO: 1). Therefore, the peptide comprising the amino acid sequence ofposition 17 to position 116 of pro line-rich protein 4 is a peptideconsisting of the amino acid sequence of SEQ ID NO: 1 or aterminal-deleted sequence thereof, wherein the terminal-deleted sequenceis a sequence:

-   1) derived from the original sequence by deletion of 1 to 16    N-terminal amino acids, and/or-   2) derived from the original sequence by deletion of 1 to 18    C-terminal amino acids, and the peptide consisting of the    terminal-deleted sequence has a neuroprotective action. Therefore,    such a peptide comprises 100 to 134 amino acid residues.

The modified sequence is a sequence derived from the original sequenceby substitution, deletion, or addition of one or several amino acids. Itis desirable that the number of amino acids substituted, deleted oradded be 1 to 3, preferably 1 or 2, and most preferably 1, so long asthe peptide has a neuroprotective action. It is desirable that an aminoacid substituted be substituted with an amino acid having the sameproperty. Moreover, the modified sequence refers to a sequence having apredetermined sequence identity to the original sequence, wherein thepeptide consisting of the modified sequence can exhibits the function oreffect of the peptide consisting of the original sequence. Thepredetermined sequence identity (BLAST) refers to a sequence identity ofat least about 75%, about 80%, about 85%, about 90%, about 95%, about97%, about 98%, or about 99%.

A peptide having a partial sequence is not limited in particular so longas the peptide has a neuroprotective action. Examples of the partialsequence include a sequence without a signal sequence, aterminal-deleted sequence without an N-terminal sequence and/or aC-terminal sequence, which does not contribute to the activity, and asequence contributing to the activity. So long as the partial sequencehas neuroprotective action, it may be of any length. For example, it mayhave an amino acid length of 4 to 133.

In another situation, in another aspect of the present invention, thepresent invention relates to a peptide consisting of the sequence ofQQRPP (SEQ ID NO: 3) or an added sequence derived therefrom by additionof amino acids to the N-terminal, C-terminal or both terminals thereof.The added sequence means a sequence derived from SEQ ID NO: 3 byaddition of one or two amino acids to N-terminal, and/or a sequencederived from SEQ ID NO: 3 by addition of one or two amino acids toC-terminal, wherein a peptide consisting of the added sequence hasneuroprotective action. Added amino acid can be of any amino acids, butit is preferable that the amino acid is an amino acid corresponding tothe amino acid sequence (SEQ ID NO: 2) of commercially availableproline-rich protein 4, or an amino acid having the same property as anamino acid corresponding to the amino acid sequence (SEQ ID NO: 2) ofcommercially available proline-rich protein 4.

Therefore, preferable peptides according to the present invention arelisted below, but this list is not intended to limit the presentinvention to these peptides.

TABLE 3 Peptide sequence QQP (SEQ ID NO: 3) QQRPPQ (SEQ ID NO: 4) QQRPNR(SEQ ID NO: 5) NQQRPP (SEQ ID NO: 6) NQQRPPQ (SEQ ID NO: 7) NQQRPPQR(SEQ ID NO: 8) QNQQRPP (SEQ ID NO: 9) QNQQRPPQ (SEQ ID NO: 10) QNQQRPPQR(SEQ ID NO: 11)

The phrase “amino acid having the same property” means an amino acidhaving a side chain of the same property. For example, amino acids thathave non-polar side chains include, for example, glycine, alanine,valine, leucine, isoleucine, methionine, proline, phenylalanine,tryptophan; amino acids that have polar side chains include, forexample, serine, threonine, asparagine, glutamine, tyrosine, cysteine;amino acids that have basic side chains include, for example, lysine,arginine, histidine; and amino acids that have acid side chainsincludes, for example, aspartic acid, glutamic acid.

Generally, the shorter the peptide, the higher its permeability acrossthe blood-brain barrier or the blood-retinal barrier, and moreover, theeasier its synthesis is. On the other hand, a certain length is requiredfrom the perspective of securing a neuroprotective action. A peptide ofthe present invention has a neuroprotective action and high in vivostability, in particular in a vitreous body. It is preferable that highin vivo stability is preferable in terms of exhibiting a neuroprotectiveaction for a long period at a small dose. Since the peptide of presentinvention has high stability in particular in a vitreous humor solution,it can have a neuroprotective action over a long period of time in avitreous body by administration through intravitreal injection.

The peptide according to the present invention can be prepared by anymanufacturing process. For example, it can be prepared by carrying out asolid-phase synthesis or a liquid-phase synthesis using a Boc method oran Fmoc method, etc. Alternatively, the peptide according to the presentinvention can be obtained by synthesizing a long-chain peptideconsisting of the sequence such as SEQ ID NO: 1 or 2, which comprisingthe peptide according to the present invention, in host cells by using agene transfer method, purifying the long-chain peptide by means of apolyhistidine tag or the like, and then, cleaving the resultinglong-chain peptide.

The peptide according to the present invention encompasses derivativesin which the N-terminal amino group, the C-terminal carboxy group or afunctional group of an amino acid side-chain has been arbitrarilymodified, so long as the neuroprotective action is not lost. Examples ofmodification include the addition of a protecting group to an aminogroup (e.g., acetylation, formylation, Boc-protection, Fmoc-protection),the esterification of a carboxyl group (e.g., ethylation), etc.Moreover, modification may include modification that normally occurs inthe living body such as phosphorylation, amidation, methylation,esterification, acetylation, etc., modification that occurs during theprocess of synthesis or facilitates purification such as biotinylation.Moreover, modification such as PEGylation may be added for the purposeof prolonging the half-life of a peptide.

The peptide according to the present invention includes apharmaceutically acceptable salt. Examples of the pharmaceuticallyacceptable salt include salts formed with an inorganic acid (e.g., ahydrochloride salt, a hydrobromide salt, a sulfate, and a phosphate) andsalts formed with an organic acid (a methanesulfonate, a benzenesulfonate, a p-toluenesulfonate, a formate, an acetate, atrifluoracetate, an oxalate, a citrate, a malonate, a fumarate, amaleate, a tartrate, a succinate, malate, etc.) or salts formed with abase (e.g., an ammonium salt, a methyl pyridinium salt, an acetylpyridinium salt, etc.). The peptide according to the present inventionalso comprises a hydrate or a solvate.

The neuroprotective agent according to the present invention refers toan agent having a neuroprotective action. Therefore, a neuroprotectiveagent can protect a nerve from damage of nerve cells, a disorderaccompanied by degeneration of nerve cells and/or cell death, and canalso be referred to as a nerve cell death (apoptosis and or necrosis)inhibiting drug, a nerve cell degeneration inhibiting drug, a nerve cellstress mitigating drug, a nerve cell toxicity resistance improvingagent, a nerve cell viability improving agent, and an abnormal proteinaccumulation inhibiting drug.

In the present specification, the term “neuroprotective action” is usedto refer to the action of protecting nerve cells from damage,degeneration and or cell death, and preferably, the term refers to theaction of protecting from nerve cell death. More specifically, the term“neuroprotective action” may include the suppression of nerve cell death(apoptosis and/or necrosis), the suppression of nerve cell degeneration,the mitigation of nerve cell stress, the improvement in resistance tonerve cell toxicity, the improvement in the survival/viability of nervecells, the suppression of abnormal protein accumulation, etc. Nervecells can be damaged by exposure to a neurotoxic substance and adeficiency in oxygen or a nutritional substance, as well as by physicaldamage, and if the damage exceeds a certain level, nerve cell death iscaused. Moreover, a nerve cell can be subjected, to degeneration as aresult of an accumulation of a neurotoxic substance, and ultimatelynerve cell death is caused. It is shown in Examples that a peptide ofthe present invention, inhibits cell death under hypoxic condition, buta peptide of the present invention does not only inhibit cell death, ithas a protective effect against damage or degeneration of nerve cells atthe stage prior to that cell-death stage. Therefore, one could say thata peptide according to the present invention has a neuroprotectiveaction against hypoxia-induced stress and nerve cell death. Neurotoxicsubstances can be mainly divided into exogenous toxic substances andendogenous toxic substances. An exogenous toxic substances includeschemical substances such as a heavy metal, alcohol, a botulinum toxin,etc. An endogenous toxic substances includes reactive oxygen species,neurotransmitter substances such as glutamate, etc., and abnormalproteins. A neuroprotective action can be easily measured by a personskilled in the relevant art. For example, after culturing nerve cellsunder various types of stress, such as a low-oxygen load, exposure to aneurotoxic substance, nutrient depletion, exposure to ultraviolet light,in culture media with the test substance (drug group) and culture mediawithout the test substance (control group); measuring the number ofviable cells and the number of dead cells in die media, and calculatingthe proportion of viable cells per total number of cells, one can judgethat a test substance has a neuroprotective action, if the proportion ofviable cells of the drag group is higher than the proportion of viablecells of the control group. In a more preferable aspect, a drag groupcan be compared to a positive control group to which a substance knownto have a neuroprotective action such as IFG-1 or NGF, etc., has beenadded, and a neuroprotective action is determined by determining whetheror nest the ding group has a protective action equivalent to or greaterthan that of the positive control group. Another example includesdetermining the neuroprotective action by carrying cart an in vivoanimal study.

The neuroprotective agent comprises at least one peptide selected fromthe peptides according to the present invention. Peptides that can beincluded in such a neuroprotective agent includes, for example but notbe limited to, peptides of sequences selected from a group consisting ofSEQ ID NOS: 1 to 11.

In another situation, the present invention also relates to apharmaceutical composition for treating or preventing nerve damage,containing a therapeutically effective dose of the abovementionedpeptide or neuroprotective agent. Nerve damage can be treated byadministering the pharmaceutical composition of the present invention toa patient having nerve damage; or nerve damage can be prevented byadministering the pharmaceutical composition of the present invention toa patient with potential nerve damage. Moreover, the term“treat”/“treatment” refers to preventing the worsening of the condition,maintaining the status quo of the condition, reducing or curing thecondition, when damage or disease has occurred; and the term“prevent”/“prevention” refers to preventing the occurance of damage ordisease before it occurs.

A peptide, neuroprotective agent or pharmaceutical composition accordingto the present invention can be used in the method of treating orpreventing nerve damage, and can be administered to a subject sufferingfrom nerve damage. Such nerve damage refers to a condition caused, bydegeneration cell death of nerve cells, in which the function of thenerve cells is damaged, and includes cerebrovascular disorders andneurodegenerative diseases.

Cerebrovascular disorders include a bleeding disorder such as a brainhemorrhage, a subarachnoid hemorrhage; and a disorder caused by anocclusion of a cerebral blood vessel such as a cerebrovascular bloodclot, a cerebral infarction, a cerebral circulatory insufficiency, etc.In both bleeding disorders and occlusive disorders, nerve cells in thebrain are in a hypoxic state, which causes cell death. Therefore, apeptide, a neuroprotective agent or a pharmaceutical compositionaccording to the present invention can be administered for a therapeuticor preventive purpose for such cerebrovascular disorders.

Neurodegenerative diseases include Degenerative brain diseases andcentral neurodegenerative diseases such as, dementia, Parkinson'sdisease, spinocerebellar degeneration, Creutzfeldt-Jakob disease,Alzheimer's disease, Huntington's chorea, multiple sclerosis, mad cowdisease, epilepsy, etc.; motor neuron degenerative diseases such asspinal progressive muscular atrophy, amyotrophic lateral sclerosis,spinal and bulbar muscular atrophy: and sensory neurodegenerativediseases. Sensory neurodegenerative diseases includes degenerativediseases of optic nerves, auditory nerves, touch sensory nerve, tastenerve, and olfactory nerve. Degenerative disease of optic nervesincludes glaucoma, retinal pigmentary degeneration, age-related maculardegeneration, diabetic retinopathy, etc., and auditory nervedegenerative diseases includes hearing loss.

A peptide, neuroprotective agent or pharmaceutical composition accordingto the present invention is provided in a dosage form suited toparenteral administration or oral administration, but from theperspective of using it as a peptide formulation, a parenteraladministration would be preferable. Examples of parenteraladministration include intravenous, intra-arterial, subcutaneous,topical, intraperitoneal, intramuscular, intranasal, percutaneous,transmucosal, intrathecal, rectal, intramuscular, intracerebral,intrathecal, subarachnoid, epidural, eye drop instillation, ear dropinstillation, nasal drop instillation, and intraocular administration.More specifically, subconjunctival administration, sub-tenonadministration, and intravitreal administration can be mentioned asroutes of intraocular administration. A neuroprotective agent accordingto the present invention can be formulated in a suitable dosage formaccording to the route of administration, for example, in any form: e.g.eye chops, an injectable formulation, powder formulation, infusionformulation, granular formulation, tablet, suppository, etc. Forparenteral administration, eye drops, injectable formulation, infusionformulation, a powder medicine for extemporaneous preparation, etc. arepreferable. Examples of intraocularly administered formulation that canbe mentioned include an intravitreally injectable formulation, asubconjunctivally injectable formulation, and a sub-tenon injectableformulation. Moreover, these pharmaceutical formulations may containvarious pharmaceutically acceptable adjuvants, i.e. a carrier or otherauxiliary agent, e.g. an additive such as a stabilizing agent, apreservative, a soothing agent, an emulsifier, etc. Moreover, it canalso be used in combination with another medicine having aneuroprotective action.

The entire contents of ail literature cited in the present specificationare incorporated herein by citation.

The purpose of the Examples of the present invention described below isonly to exemplify the present invention, and they do not limit thetechnical scope of the present invention. The technical scope of thepresent invention shall be limited by the wording of the claims alone.The present invention may be changed on condition that such change doesnot depart from the spirit of the present invention: For example, anaddition or substitution to or a deletion of a constituent feature ofthe invention can be realized.

EXAMPLES Test Example 1: Synthesis of Peptides

Physiologically active peptides of the present invention, which wereused in tests described below, were solid-phase synthesized by an Fmocmethod using a peptide synthesizer (model: PSSM-8 manufactured byShimadzu Corporation). The synthesized peptides were analyzed by massspectrometry (MALDI TOF), and as shown in the table below, themeasurement values were highly consistent with the theoretical values.

TABLE 4 Mass  spectrometry Theoretical Measurement value value SequencePeptide 1 1636.8 1636.6 HPPPPPFQNQQRPP (SEQ ID NO: 12) Peptide 2 1286.41286.5 HPPPPPFQNQQ (SEQ ID NO: 13) Peptide 3 916.0 916.6 HPPPPPFQ(SEQ ID NO: 14) Peptide 4 624.7 625.2 QQRPP (SEQ ID NO: 3)

Test Example 2: The Protective Effect of the Peptide Against Cell Deathof Human Neuroblastoma (SH-SY5Y Cells) from Hypoxia

1. Cell Culture

SH-SY5Y cells (ATCC) were sustainably cultured in a DMEM/F12 medium(Invitrogen, 11330-032) supplemented with a 10% serum (Invitrogen,10062-147) under the condition of 37° C. and 5% CO₂.

2. Hypoxia-Induced Cell Death Assay

The cells were seeded on a 96-well plate (Iwaki, 3860-096) at a densityof 2×10⁴ cells/100 μl/well. The following day the medium was replacedwith a DMEM/F12 medium comprising a 1% serum and 10 μM of a retinoicacid (Wako, 302-79-4) and differentiated by incubation for 2 days. Afterreplacing the medium with a glucose-free DMEM medium supplemented with100 nM of Peptide 1, a hypoxia state (O₂ concentration of 0.1% or less)was induced by putting the plate in an Anaero Pack rectangular jar withAnaero Pack Kenki 5% (Mitsubishi Gas Chemical Company, Inc., A-07) withhermetically sealing, and culturing at 37° C. Alter culturing for 24hours in the hypoxia state, the cell-death inhibiting rate by thepeptide was calculated by measuring the amount of LDH in the cells andthe medium, using an LDH cytotoxicity detection kit (Takara, MK401).Moreover, 100 nM of an IGF-1 was used as a positive control in theexperiment IGF-1 has a protective effect against nerve cell death (Mol.Cell Neurosci, 2011, 47 (3), 181-190).3. Test Results

As shown in the table below, 100 nM of Peptide 1 significantlysuppressed the cell death from hypoxia of the neuroblastoma SH-SY5Ycells. From this result, if was ascertained that Peptide 1, which is apartial sequence of proline-rich-protein 4 identified in tears, exhibitsa neuroprotective action. The rate of suppression of cell death washigher than the rate detected for IGF-1, an existing nerve growthfactor.

TABLE 5 Cell lethality (%) Peptide 1 (100 nM) 86.1 ± 43.9* IGF-1 (100nM) 88.1 ± 8.3*  Vehicle  0.0 ± 16.5* The mean ± standard error (n = 3)*p < 0.05 (compared to the vehicle. t-test)

Test Example 3: The Protective Effect of the Peptide Against Cell Deathof Rat Retinal Ganglion Cells (RGC) from Hypoxia

1. Preparation of the Rat Retinal Ganglion Cells

Retinal ganglion cells were isolated by a magnetic activated cellsorting method (MACS). Retinas were harvested from neonatal rats (Slc:Sprague-Dawley, 7-day old) and the tissue was dissociated using a NeuralTissue Dissociation Kit (Miltenyi Biotec, 130-094-802). Then, using aRetinal Ganglion Cell Isolation Kit (Miltenyi Biotec, 130-096-209), theendothelial cells and microglia were removed, and a positive selectionfor CD90.1⁺ RGC was carried out by means of CD90.1MicroBeads. The testwas carried out with the approval of the Animal Test Ethics Committeebased on the Act on Welfare and Management of Animals (Act number 105 ofOct. 1, 1973, last revision: Act number 38 of Jun. 12, 2013), etc.2. Cell Culture

After suspending the isolated RGC in a neurobasal medium (GibcoCorporation, 12348-017) containing a B27 supplement (Invitrogen,0080085-SA), 1 mM of L-glutamine (Gibco, 25030), 50 ng/ml of BDNF(Peprotech, 25-02), 50 ng/ml of CNTF (Peprotech, 450-50) and 5 μm ofForskolin. (Sigma, F6886), they were seeded on a 96-well plate (Corning,354596), ‘which’ was coated with poly-D-lysine/laminin, at a density of5×10³ cells/100 μl/well, and incubated at 37° C. and 5% CO₂ for threedays.

3. Hypoxia-Induced Cell Death Assay

After having replaced the media with neurobasal media comprising a B27supplement, 1 mM of L-glutamine 50 and each peptide, a hypoxia state (O₂concentration of 0.1% or less) was induced by putting the plate in anAnaero Pack rectangular jar with Anaero Pack Kenki 5% (Mitsubishi GasChemical Company, Inc., A-07) with hermetically sealing, and incubatingat 37° C. After incubating for 24 hours under hypoxia condition,Calcein-AM and Ethidium homodimer (Invitrogen, L3224) were added to themedia. As a result of staining the cells, it was possible to distinguishbetween the viable cells and the dead cells. The number of viable cellsand dead cells were measured using an Image Pro plus, and the rate ofviability was given as “the proportion of the viable cell count relativeto the total cell count.”4. Test Results

As shown in the table below, Peptides 1 to 4 significantly suppressedthe cell death from hypoxia. These results suggest that Peptide 4 is thepeptide with the shortest active sequence.

TABLE 6 Rate of cell-death suppression (%) Peptide 1 (100 nM) 25.4 ±7.8* Peptide 2 (100 nM) 26.4 ± 4.8* Peptide 3 (100 nM)  27.2 ± 6.6**Peptide 4 (100 nM)  31.1 ± 7.8** Vehicle 0.0 ± 7.4 The mean value ±standard error (n = 14-19) *p < 0.05 **p < 0.01 (compared to thevehicle. t-test)

Test Example 4: The Protective Effect of Proline-Rich Protein 4 (PRP4)Against Cell Death of Rat Retinal Ganglion Cells (RGC) from Hypoxia

1. Preparation of the Rat Retinal Ganglion Cells and Cell Incubation

The isolation and incubation of RGC were performed by the same methodsas for Test Example 3-1, and 2.

2. Hypoxia-Induced Cell Death Assay

Commercially available PRP4 (Abnova, H0011272-Q01) having the sequenceof SEQ ID NO: 2, was used for Peptide 5. The evaluation of Peptide 5 wascarried out by the same evaluation method as used for Test Example 3-3.

3. Test Results

As shown in the table below. Peptide 5 significantly suppressed the celldeath from hypoxia.

TABLE 7 Rate of cell-death suppression (%) Peptide 5 (10 nM) 66.2 ±14.4* Vehicle 0.0 ± 21.9 The mean value ± standard error (n = 12 to 18)*p < 0.05 (compared to the vehicle. t-test)

Test Example 5: Test of the Stability of Peptide 1 in a Vitreous HumorSolution

(Two) rabbits were euthanized by intravenous administration of 5 ml of apentobarbital sodium solution, and then their eyeballs were removed. Anincision was made in the equatorial region of each eyeball and thevitreous humor-solution was harvested from each eyeball using a syringe(18G injection needle) at the site of incision. The mixed vitreous humorsolution was filtered with a cell strainer (40 μm) and the filtrate wasused as the vitreous humor solution for the stability tests. 600 μl ofthe vitreous humor solution was mixed with 1.2 μl of a Primocin at aconcentration of 50 mg/ml and the resultant solution was used as samplesolution. 270 μl of the sample solution and 1 of Peptide 1 in a 30 μlaqueous solution were mixed and the resultant mixture was used as thetest sample. The test sample was incubated at 37° C. 7 days later, 30 μlof the test sample was collected and mixed with 200 μl of a 1 N aceticacid. The resultant mixture was boiled at 100° C. for 10 minutes andthat mixture was then used for the analysis samples. The analysissamples were stored frozen at −20° C. until it was time to take readingsusing them. After they were melted, they were put through a centrifugaltreatment (15000×g) at 4° C. for 20 minutes. Then, the concentration ofPeptide 1 in each sample was analyzed using liquid chromatography. Theconcentrations were calculated from the area values obtained using theexternal standard method. The conditions of analysis of the liquidchromatography were as outlined below.<Conditions of Analysis of the Liquid Chromatography>

Mobile phase A: 10% acetonitrile + 0.1% trifluoro acetic acid Mobilephase B: 60% acetonitrile + 0.095% trifluoro acetic acid Gradientanalysis: B conc. 0% to 50% 15 minutes Flow rate: 1 ml/minute Column:YMC-Pack ODS-AQ S-3 μm, 12 nm 150 × 4.6 mm I.D. AQ12S03-1546WT System:Shimadzu LC-20A Column oven: 40° C.

The residual rate of Peptide 1 after 7 days' incubation with thevitreous humor solution is shown in Table 9. The residual rate wascalculated using the following formula:Residual rate=(the concentration of peptide after 7 days' incubation/theconcentration of peptide in the test samples immediately after they wereprepared)×100  [Formula 1]

TABLE 8 Residual ratio (%) Peptide 1 98.4This result established the fact that Peptide 1 is stable in a vitreoushumor solution.Formulation ExamplesA pharmaceutical product containing a peptide according to the presentinvention as an active ingredient can fee manufactured by formulationssuch as the following for example.1. Capsule Medicine

(1) Peptide 1 40 mg (2) lactose 70 mg (3) microcrystalline cellulose 9mg (4) magnesium stearate 1 mg 1 capsule 120 mgMix the fall amounts of (1), (2), and (3) and half the amount of (4) andthen granulate the mixture. Add the remaining amount of (4) to thegranulated mixture and encapsulate the whole resultant mixture in agelatin capsule.2. Tablet

(1) Peptide 4 40 mg (2) lactose 58 mg (3) corn starch 18 mg (4)microcrystalline cellulose 3.5 mg (5) magnesium stearate 0.5 mg 1 tablet120 mgMix the fall amounts of (1), (2), and (3), two thirds of (4) and half of(5) then granulate the mixture. Add the remaining amounts of (4) and (5)to the granulated mixture and compress the resultant mixture into atablet form.2. Vitreous Humor Injection SolutionContent of 1 ml

(1) Peptide 1 40 mg (2) purified white sugar 50 mg (3) sodium chloride2.34 mg (4) polysorbate 80 q.s. (5) disodium hydrogen phosphate q.s. (6)sodium dihydrogen phosphate q.s. (7) sterilized purified water q.s.Prepare a vitreous humor injection solution by dissolving ingredients(1) to (6) in the sterilized purified water (7).

The invention claimed is:
 1. A peptide consisting of: (a) the amino acidsequence of QQRPP (SEQ ID NO: 3) or a pharmaceutically acceptable saltthereof, or (b) a sequence derived therefrom by addition of one or twoamino acids to N-terminal, C-terminal or both terminals thereof, whereinthe amino acids added to N-terminal are glutamine-asparagine- orasparagine-, and the amino acids added to C-terminal are-glutamine-arginine or glutamine, or (c) a derivative of (a) or (b)wherein the N-terminal amino group, the C-terminal carboxyl group or afunction group of an amino acid side chain includes a modification, andwherein the peptide has a neuroprotective action.
 2. The peptideaccording to claim 1, wherein the modification of the derivative of (a)or (b) includes one or more modification selected from the groupconsisting of an addition of a protecting group to an amino group, anesterification of a carboxyl group, a phosphorylation, an amidation, amethylation, an esterification other than the carboxyl group, anacetylation, biotinylation and PEGylation.
 3. A peptide consisting ofthe sequence of QQRPP (SEQ ID NO:3).